Comparison of Different Promoter Methylation Assays in Breast Cancer

Karijn P M Suijkerbuijk,Xiaojuan Pan,Paul J Van Diest,Elsken Van Der Wall,Marc Vooijs

doi:10.1155/2010/185219

Abstract

Background: Promoter hypermethylation has emerged as a promising cancer biomarker. Currently, a large variety of quantitative and non-quantitative techniques is used to measure methylation in clinical specimens. Here we directly compared three commonly used methylation assays and assessed the influence of tissue fixation, target sequence location and the amount of DNA on their performance.Methods: We used Methylation-Specific PCR (MSP), Quantitative Multiplex MSP (QM-MSP) and Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) to compare methylation of CCND2, SCGB3A1, RARB and RASSF1 on DNA from 40 breast carcinomas.Results: A comparison between MSP and QM-MSP on the same samples showed a high discrepancy: 20% of tumors that showed no methylation in MSP gave >10% methylation in QM-MSP. In contrast, QM-MSP correlated strongly with MS-MLPA when targeting the same sequence in DNA from paraffin embedded as well as fresh frozen tissue. This correlation declined when target sequences were non-overlapping. In titration experiments, MSP and MS-MLPA performed robust with 10 ng of DNA, while QM-MSP was at least ten-fold more sensitive.Conclusion: Despite the difference in molecular basis, QM-MSP and MS-MLPA showed moderate to strong correlations. In contrast, there was a poor concordance between either of these techniques and non-quantitative MSP. For biological samples with scarce DNA, QM-MSP is the method of choice.

Highlights

Over the last decade, the importance of epigenetic silencing of tumor suppressor genes by DNA promoter hypermethylation has been recognized as a common mechanism driving carcinogenesis [10]
To do so we compared DNA methylation frequencies of 4 commonly methylated genes in DNA isolated from 40 paraffinembedded invasive human breast cancer specimens measured by Methylation-Specific Polymerase Chain Reaction (PCR) (MSP), Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) and Quantitative Multiplex MSP (QM-MSP)
The non-quantitative assay MSP showed highly discrepant results when compared with the quantitative QM-MSP assay (Fig. 1)

Summary

Introduction

The importance of epigenetic silencing of tumor suppressor genes by DNA promoter hypermethylation has been recognized as a common mechanism driving carcinogenesis [10]. Methylation has proven to be a promising predictive and prognostic biomarker [19], but is especially promising as a biomarker for early cancer detection in biological fluids [21] for various reasons. Methylation changes have been demonstrated in non-cancerous cells adjacent to the tumor [26]. This so-called field defect facilitates detection in heterogeneous biological samples. Methylation detection is applicable to biological fluids that contain low amounts of DNA [3,7,22]. We directly compared three commonly used methylation assays and assessed the influence of tissue fixation, target sequence location and the amount of DNA on their performance

Methods

Results

Conclusion